PTEN/MMAC1 is a tumor suppressor that is mutated in many forms of cancer. PTEN encodes a phosphatase that recognizes the important second messenger, phosphatidylinositol-3,4,5-triphosphate (PIP3,4,5), and removes the 3'-phosphate from the inositiol ring. PTEN therefore antagonizes phosphatidylinositol (PI)-3 kinase, which phosphorylates the inositol ring at the same position. Although PI-3 kinase and PTEN potentially influence many signaling pathways, perhaps the best understood pathway species, PTEN acts downstream of the insulin receptor and PI-3 kinase and upstream of AKT. Through the phosphorylation of substrates, AKT regulates many targets involved in transcription, translation, the cell cycle, and apoptosis. To test the hypothesis that PTEN regulates gene expression, they developed an adenoviral system that expressed PTEN within two hours of infection in PTEN-/- tumor cells. PTEN expression was associated with a rapid inhibition of AKT. Labeled RNAs from different times of infection were hybridized to oligonucleotide chips representing over 40,000 genes. By comparing the level of expression between uninfected, Adeno-beta-galactosidase, and Adeno-PTEN infected cells, they identified 32 candidates that were induced at least threefold within 3 hours of infection. They prepared probes for 24 of the candidates, of which 8 demonstrated PTEN-specific induction on Northern blots. The most induced gene was the insulin receptor substrate (IRS)-2. Induction of IRS-2 was confirmed at the protein level. IRS-2 is a member of a family of IRS genes that function as adaptor proteins for the transmission of insulin, IGF, and cytokine receptor signals. The major role of IRS-2 is to link an activated receptor to PI-3 kinase. In PTEN infected cells, the induced IRS-2 coimmunoprecipitated with PI-3 kinase. Thus, their preliminary data suggests that the expression of PTEN induced a feedback-loop to activated PI-3 kinase. This grant application will attempt to explore the mechanism through which PTEN regulates IRS-2 and define the role of IRS-2 in tumorigenesis. They will use recombinant adenoviruses and pharmacological agents to identify the mode through which cells are able to induce IRS2 in response to PTEN. This will include the use of adenoviruses that express PTEN mutations, dominant-negative AKT and dominant active FKHR, GSK-3, and RAF. Inhibitors of PI-3 kinase, MEK, and mTOR will also be tested. They will not only analyze these agents for their ability to induce IRS-2, they will also compare them to PTEN regarding their effect on AKT, MAPK, GSK-3, and S6 kinase activity. The p85 and p110 subunits of PI-3 kinase and AKT are potent oncogenes. To determine whether IRS-2 is altered genetically in cancer they will screen for mutations and implications in cells lines and primary tumors containing wild type PTEN. Because IRS-2 functions as an adapter protein in the transduction of insulin signals, they will investigate the effect of PTEN on insulin signaling in mice. Finally, PTEN heterozygous mice have tumors in multiple organ systems. They will breed PTEN+/- mice with IRS-2+/- mice to see if IRS-2 loss is able to attenuate the tumor phenotype.